Production of pradimicin antibiotics

ABSTRACT

The present invention relates to a fermentation process for producing BMY-28960 and desxylosyl BMY-28960, and to a novel BMY-28960-producing organism belonging to the genus Actinomadura and designated as strain AB 1236 (ATCC 55208).

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a fermentation process for theproduction of pradimicin antibiotics, and to the producing microorganismof said antibiotics. 2. Background Art

Among the various reported members of the pradimicin family produced byActinomadura, pradimicins FA-1 (Ia) and FA-2 (Ib), disclosed in U.S.Pat. No. 4,973,673, contain a D-serine moiety. Benanomicin A (II), acompound closely related to the pradimicins, has been reported in J.Antibiot., 1988, 41:807-811; it differs from the pradimicins in lackingthe sugar amino group of the pradimicins. European Patent Application432,527 published Jun. 19, 1991 discloses the compound4'-deamino-4'-axial-hydroxypradimicin FA-2 (III, hereinafter referred toas BMY-28960) which was prepared from pradimicin FA-2 by chemical means.Desxylosyl BMY-28960 is also generically disclosed in EP 432,527 and maybe prepared from desxylosyl pradimicin FA-2. ##STR1##

The chemical processes for preparing BMY-28960 and its desxylosylderivative are difficult and laborious, and produce the products in lowyield. Thus, an alternative process suitable for mass production ofthese antibiotics is highly desirable. As a result of an intensivesearch for microorganisms capable of producing BMY-28960 and desxylosylBMY-28960, a novel microorganism strain belonging to the genusActinomadura was found to be such an antibiotic producer.

SUMMARY OF THE INVENTION

The present invention provides a process for the preparation of anantibiotic of the formula ##STR2## wherein R is hydrogen or β-D-xylosyl,which comprises cultivating a strain of Actinomadura capable ofproducing said antibiotic in an aqueous medium containing an assimilablesource of carbon, nitrogen, and D-serine under aerobic conditions, andrecovering said antibiotic from the cultured broth. Preferably theproducing organism is Actinomadura sp. AB 1236, ATCC 55208. In apreferred embodiment, the fermentation medium further containsD-cycloserine.

In another aspect the present invention provides a biologically pureculture of the microorganism Actinomadura sp. AB 1236 having theidentifying characteristics of ATCC 55208 and capable of producingBMY-28960 and desxylosyl BMY-28960 upon cultivation in an aqueous mediumcontaining an assimilable source of carbon, nitrogen, and D-serine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an IR spectrum (KBr) of BMY-28960.

FIG. 2 is a ¹ H NMR spectrum (DMSO-d₆, 400 MHz) of BMY-28960.

FIG. 3 is a ¹³ C NMR spectrum (DMSO-d₆, 100 MHz) of BMY-28960.

FIG. 4 is an IR spectrum (KBr) of desxylosyl BMY-28960.

FIG. 5 is a ¹ H NMR spectrum (DMSO-d₆, 400 MHz) of desxylosyl BMY-28960.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a fermentation process suitable for massproduction of BMY-28960 and desxylosyl BMY-28960. The process utilizes anovel microorgansim belonging to the genus Actinomadura.

I. Screeninq of BMY-28960-Producing Microoragnisms

A loopful of each actinomycete isolated from soil samples was inoculatedas a patch onto three agar plates, namely glucose-yeast-soytone agar(glucose 1%, yeast extract 0.05%, soytone (Difco Laboratories) 0.05%,CaCl₂.2H₂ O 0.01% and agar 1.5%), glycerol-yeast-soytone agar (glycerol1%, yeast extract 0.05%, soytone 0.05%, CaCl₂.2H₂ O 0.01% and agar 1.5%)and starch-yeast-soytone agar (soluble starch 1%, yeast extract 0.05%,soytone 0.05%, CaCl₂.2H₂ O 0.01% and agar 1.5%), and then incubated at37° C. for 1 to 2 weeks. Strains that produced dark pink to dark reddiffusible pigments in each agar plate were inoculated into 500-mlErlenmeyer flasks containing 100 ml of a production medium composed ofglycerol 2%, Esusan mito (Ajinomoto Co., Ltd.) 1.5%, CoCl₂.6H₂ O0.0001%, KH₂ PO₄ 0.1125%, K₂ HPO₄ 0.0025% and D-serine 0.2%, andincubated with rotary shaking (200 rpm) at 32° C. After 10 days ofincubation, production of BMY-28960 in the broth was monitored by theagar well assay of the supernatant using Candida albicans A9540 as testorganism. The broths showing anti-Candida activity were centrifuged,diluted 10 fold with DMSO, and filtered (Gelman Sciences Japan, Ltd.,Ekicrodisc 13CR, Pore size: 0.45 μm). The filtrates were analyzed byHPLC on Excel pak SIL-C185R (Yokogawa Electronic Co., Ltd.) usingacetonitrile: 0.02 M phosphate buffer, pH 7.0 (15:85), at a flow rate of1 ml/min. with 254 nm detection and by TLC on silica gel thin layerplates (Kiesel gel 60 F254 0.25 mm; mfd, Merck). The developing solventsystems used were n-butanol:acetic acid:water (2:1:1BW-14) and methylacetate:n-propanol:28% aq. ammonia (45:105:60, S-114). BMY-28960 has anHPLC retention time of 11.5 min. with the above solvent system and Rfvalues of 0.50 and 0.24 using BW-14 and S-114, respectively.

Various actinomycetes were screened and strain AB 1236 belonging to thegenus Actinomadura was found to produce the desired compound at a levelsuitable for mass production. Taxonomical characteristics of strain AB1236 will be described below.

II. BMY-28960-Producing Organism

Actinomadura sp. AB 1236 is a new strain isolated from a soil samplecollected at Shinjuku, Tokyo, Japan on Oct. 24, 1990. A culture ofstrain AB 1236 has been deposited with the American Type CultureCollection under the BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OFTHE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE, andall restrictions on the availability to the public of the depositedmicroorganism will be irrevocably removed upon the granting of a patentfrom this application. The deposited culture has been assigned theaccession number ATCC 55208.

A. Morphology and cultural characteristics of strain AB 1236

The media and procedures used for the taxonomic study of the strain werethose described by Shirling and Gottlieb in "Methods forCharacterization of Streptomyces species", Int. J. Syst. Bact., 1966,16:313-340; by Waksman in The Actinomycetes, Vol. II, "Classification,Identification and Description of Genera and Species, pp. 328-334, publ.by The Williams and Wilkins Co., Baltimore, 1961; and by Arai in CultureMedia for Actinomycetes, publ. by The Society for Actinomycetes Japan,1975. The strain was incubated at 37° C. for 2 to 4 weeks. Colordetermination was made by comparing the color of the culture with colorchips according to the Manual of Color Names (Japan Color EnterpriseCo., Ltd., 1987).

Strain AB 1236 grew better on organic media than on inorganic media attemperatures between 20° and 41° C. and formed a branched vegetativemycelium. The color of mature aerial mycelia was white to grayish whiteon both yeast starch agar and yeast-starch-malt agar (YSM, solublestarch 1%, yeast extract 0.1%, malt extract 0.1%, CaCl₂.2H₂ O 0.05% andagar 1.5%). Under light and scanning electron microscopes, the top ofshort sporophores had 2 to 8 spores per single chain. The shape of thesespores was subglobose (0.8-1.0×1.0-1.2 μm in size) and their surface wassmooth. These spores were not motile. The color of vegetative myceliaand diffusible pigments on organic agar media ranged from soft pink todark red. The color changed from soft orange to strong yellowish orangeby the addition of 0.1 N HCl. The cultural characteristics of strain AB1236 on various agar media are summarized in Table 1.

                  TABLE 1                                                         ______________________________________                                        Cultural characteristics of strain AB 1236                                                                 Aerial  Soluble                                  Medium   Growth    Reverse   mycelium                                                                              pigment                                  ______________________________________                                        Sucrose  Yellowish Yellowish None    None                                     nitrate agar                                                                           white (393)                                                                             white (393)                                                (Waksman                                                                      med. No. 1)                                                                   Glycerol Grayish   Grayish   None    Pinkish                                  nitrate  red (60), red (60)          white (391)                              agar     Good                                                                 Glucose  Yellowish Yellowish None    None                                     asparagine                                                                             white (393),                                                                            white (393)                                                agar     Good                                                                 (Waksman                                                                      med. No. 2)                                                                   Yeast ext.-                                                                            Dark red  Dark red  Grayish Dark red                                 malt ext. agar                                                                         (57), Good                                                                              (57)      white (390)                                                                           (58)                                     (ISP med.                    to pinkish                                       No. 2)                       white (391)                                      Oat meal Soft pink Soft pink White   Soft pink                                agar (ISP                                                                              (25), Good                                                                              (26)      (388),  (26)                                     med. No. 3)                  Cottony                                          Inorganic                                                                              Yellowish Yellowish None    None                                     salts-starch                                                                           white (393),                                                                            white (393)                                                agar (ISP                                                                              Good      to pinkish                                                 med. No. 4)        white (391)                                                Glycerol Dark red  Dark red  None    Soft pink                                asparagine                                                                             (58), Good                                                                              (58)              (25)                                     agar (ISP                                                                     med. No. 5)                                                                   Tyrosine agar                                                                          Dark red  Dark red  None    Soft pink                                (ISP med.                                                                              (58), Good                                                                              (58)              (25)                                     No. 7)                                                                        Nutrient agar                                                                          Yellowish Yellowish None    None                                     (Waksman white (393),                                                                            white (393)                                                med. No. 14)                                                                           Poor                                                                 Yeast starch                                                                           Dark gray-                                                                              Dark gray-                                                                              White (388)                                                                           Deep                                     agar     ish red   ish red   to grayish                                                                            yellowish                                         (61), Good                                                                              (61)      white (390),                                                                          red (53)                                                              Cottony                                          Bennett's                                                                              Dark red  Dark red  Grayish Deep pink                                agar     (57), Good                                                                              (57)      white (390),                                                                          (22)                                                                  Scant                                            ______________________________________                                    

B. Physiological characteristics of strain AB 1236

The physiological characteristics and the pattern of carbon sourceutilization of strain AB 1236 are shown in Tables 2 and 3, respectively.

                  TABLE 2                                                         ______________________________________                                        Physiological characteristics of strain AB 1236                               Test                       Results                                            ______________________________________                                        Starch hydrolysis (ISP med. No. 4)                                                                       +                                                  Nitrate reduction (Difco, nitrate broth)                                                                 -                                                  10% skimmed milk (Difco, 10% skimmed milk)                                    Coagulation                +                                                  Peptonization              -                                                  Cellulose decomposition (sucrose nitrate solution                                                        -                                                  with a strip of paper as the sole carbon source)                              Gelatin liquefaction       No growth                                          Melanine formation On ISP med. No. 7                                                                     -                                                  Temperature range for growth (°C.)                                                                20-41                                              Optimum temperature (°C.) (on Yeast starch agar)                                                  30.5-35.5                                          pH range for growth        6-8                                                Optimum pH (On trypticase soy broth, BBL)                                                                7                                                  ______________________________________                                         -: Negative                                                                   +: Positive                                                              

                  TABLE 3                                                         ______________________________________                                        Utilization of carbon sources by strain AB 1236                               Carbon source   Utilization                                                   ______________________________________                                        D-Glucose       +                                                             L-Arabinose     +                                                             D-Xylose        +                                                             Inositol        +                                                             Mannitol        +                                                             D-Fructose      +                                                             L-Rhamnose      +                                                             Sucrose         +                                                             Raffinose       +                                                             ______________________________________                                         +: Positive                                                                   (ISP med. No. 9, 37° C. for 3 weeks)                              

Antibiotic susceptibility of strain AB 1236 was tested using antibioticdisks (Tridisk, Eiken Chemical Co., Ltd.). The disks were placed ontothe surface of yeast-glucose-malt agar (yeast extract 0.1%, glucose 1%,malt extract 0.1%, CaCl₂.2H₂ O 0.05% and agar 1.5%) which had beenseeded by strain AB 1236 (4% inoculum), and the plates were thenincubated at 37° C. for 4 days. Strain AB 1236 was resistant to 50 μg offosfomycin and 300 U of polymixin B, and susceptible to 20 U ofampicillin, 1 μg of clavulanic acid, 2 μg of ticarcillin, 10 μg ofcephalexin, 30 μg of tetracycline, 10 μg of chloramphenicol, 0.5 μg oferythromycin, 2 μg of josamycin, 2 μg of lincomycin, 5 μg of kanamycin,5 μg of gentamicin, 5 μg of tobramycin, 2 μg of nalidixic acid, 2 μg ofnorfloxacin and 50 U of colistin.

C. Chemical analysis of AB 1236 cells

Whole-cell compositions were analyzed by the method described by Beckerand Lechevalier in "Rapid Differentiation between Nocardia andStreptomyces by Paper Chromatography of Whole Cell Hydrolysate", Appl.Microb., 1964, 12:421-423, and in "Chemical Compositions of Cell-WallPreparations from Strains of Various Form-Genera of AerobicActinomycetes", Appl. Microb., 1965, 13:236-243. Strain AB 1236contained meso-diaminopimeric acid, madurose, ribose, mannose, glucose,and galactose. Thus, strain AB 1236 has a cell wall belonging to typeIII B. Mycolic acids were not detected by the method of Minnikin et alin "Differentiation of Mycobacterium, Nocardia, and Related Taxa byThin-Layer Chromatographic Analysis of Whole-Organism Methanolysates",J. Gen. Microb., 1975, 88:200-204. Phospholipid analysis using theprocedure of Lechevalier et al. in "Identification of AerobicActinomycetes of Clinical Importance", J. Lab. Clin. Med., 1968,71:934-944, and in "Chemotaxonomy of Aerobic Actinomycetes: PhospholipidComposition", Biochem. Syst. Ecol.,1977, 5:249-260, showed that the cellwall of strain AB 1236 had a type P1 pattern containingdiphosphatidylinositol mannoside, phosphatidylinositol anddiphosphatidylglycerol. Analysis of the menaquinone composition usingthe procedure of Collins et al. in "A Note on the Separation of NaturalMixtures of Bacterial Menaquinones Using Reverse-Phase Thin-LayerChromatography", J. Appl. Bacteriol., 1980, 48:277-282, revealed 47%MK-9 (H8), 35% MK-9 (H6), 10% MK-9 (H4) and 8% MK-9 (H10). Thewhole-cell fatty acids determined by the method of Suzuki et al. in"Taxonomic Significance of Cellular Fatty Acid Composition in SomeCoryneform Bacteria", Int. J. Syst. Bacteriol., 1983, 33:188-200,consisted of 49% 14-methylpentadecanoic acid (iso 16:0), 13%14-methylhexadecanoic acid (anteiso-17:0) and 8% 10-methylheptadecanoicacid (10Me-17:0), and other minor fatty acids.

Strain AB 1236 has morphological, cultural, and chemotaxonomicproperties that are consistent with those of the genus ActinomaduraLechevalier and Lechvalier, and with the definition of this genusproposed by Kroppenstedt et al. in "Taxonomic Revision of theActinomycete Genera Actinomadura and Microtetraspora", System. Appl.Microbiol., 1990, 13:148-160. Thus, strain AB 1236 has been identifiedas a species of Actinomadura.

III. Antibiotic Production

BMY-28960 and desxylosyl BMY-28960 may be produced by strain AB 1236under conditions conventionally used for producing common fermentationproducts. The producing organism is grown in a nutrient mediumcontaining an assimilable source of D-serine in addition to knownnutritional sources for actinomycetes, i.e. assimilable sources ofcarbon and nitrogen plus optional inorganic salts and other known growthfactors. Submerged aerobic conditions are preferably employed for theproduction of large quantities of antibiotic, although for production oflimited amounts, surface cultures and bottles may also be used.

As an assimilable source of D-serine, either D-serine or DL-serine maybe used. Examples of assimilable source of carbon are glycerol; sugarssuch as ribose, glucose, sucrose, cellobiose; starch; and othercarbohydrates such as dextran. Examples of assimilable nitrogen sourceare ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodiumnitrate, and organic nitrogen sources such as peptone, meat extract,yeast extract, corn steep liquor, soybean powder, cotton seed flour andthe like. There may also be added if necessary inorganic salts such ascobalt chloride and potassium phosphate. Furthermore, the production ofantibiotic is enhanced with the addition of threonine to the productionmedium; threonine may be D-threonine, L-threonine or a mixture thereof.Addition of D-cycloserine thereto further improves antibioticproduction. A preferred liquid medium is the one described in Example 2or Example 3. Another more conventional liquid medium is composed ofglucose and/or glycerol (1-4%), Pharmamedia (1-3%), KH₂ PO₄ (0.1-0.2%),and D-serine (0.1-0.2%). Adekanol, silicone and the like can be used asantifoaming agents.

Production of the antibiotic may be carried out at any temperatureconducive to satisfactory growth of the producing organism. Ordinarily,optimum antibiotic production is obtained in shake flasks after anincubation period of 5-14 days, although a longer period may benecessary in certain cases. Aeration in shake flasks is achieved byagitation, e.g. shaking on a rotary shaker. If fermentation is to becarried out in tank fermentors, it is desirable to produce a vegetativeinoculum in a nutrient broth by inoculating the broth culture from aslant culture or a lyophilized culture of the organism. After obtainingan active inoculum in this manner, it is aseptically transferred to thefermetation tank medium. Agitation in the tank fermentor is provided bystirring and aeration may be achieved by injection of air or oxygen intothe agitated mixture. Antibiotic production may be monitored usingchromatographic or spectroscopic techniques, or by a conventionalbiological assay. Preferred fermentation conditions are aerobiccultivations at pH 5-8 at 20°-37° C. for 5-14 days, preferably at pH 6-7at 28°-35° C. for 5-12 days.

Although the present invention describes the production of antibiotic bya specific strain of microorganism, it is widely known that thetaxonomic properties of Actinomycetes may be varied naturally orartificially. Thus, it is to be understood that the process of thepresent invention is not limited to the particular organism mentioned,but includes variants and mutants derived from the particular strain byvarious artificial methods such as ultraviolet light or X-rayirradiation, or by chemical mutagenic agents such asN-methyl-N'-nitro-N-nitrosoguanidine. Mutants and variants so producedmay be screened for antibiotic production by the procedure describedearlier in the present disclosure.

IV. Isolation and Purification of the Antibiotic

BMY-28960 and desxylosyl BMY-28960 may be isolated from cultured brothsby conventional procedures for isolating hydrophilic acidic substances.Examples of such procedures include organic solvent extraction, ionexchange resin, partition chromatography, and acidic precipitation;these may be used either alone or in combination. An illustrativeisolation and purification procedure follows. After completion of thefermentation, the broth is adjusted to pH 2.0 and centrifuged orfiltered. The resulting supernatant or filtrate is adsorbed on highporous polymer resin such as Diaion HP-20 (Mitsubishi Kasei Co.) andeluted with water miscible organic solvents such as methanol or acetone.The eluate thus obtained is concentrated and lyophilized to yield acrude antibiotic complex. For further purification, the crude materialmay be applied to a reversed phase silica gel column such as YMC-ODS A60(Yamamura Chemical Lab.) and eluted with acetonitrile:0.02 M phosphatebuffer, pH 7.0. Fractions containing BMY-28960 are pooled and desaltedto provide pure BMY-28960. Desxylosyl BMY-28960 may be obtained by asimilar isolating and purification procedure; preferably, thefermentation broth is not acidified.

V. Physico-chemical Properties of the Antibiotic

BMY-28960 obtained by the process of this invention has the followingphysico-chemical properties which are identical with those of BMY-28960obtained by semisynthesis as disclosed in EP 432,527.

(1) Appearance: Amorphous deep reddish orange powder

(2) Melting point:>220° C. (grad. dec.)

(3) FAB-MS (positive): m/z 844(M+H)⁺

(4) Molecular formula: C₃₉ H₄₁ NO₂₀

(5) UV absorption spectrum, λmax nm(ε): 0.02 N NaOH:MeOH(1:1):211(34,700), 320(15,100) 498(14,100)

(6) IR spectrum: as shown in FIG. 1

(7) Solubility in solvent

Soluble: Dimethyl sulfoxide, N,N-dimethylformamide and alkaline water

Slightly soluble: Ethanol, methanol and acetone

Isoluble: Ethyl acetate, benzene, chloroform, acidic water, etc.

(8) Thin layer chromatography (Silica gel plate):

Rf=0.24 (methyl acetate:n-propanol:28% agueous ammonia=45:105:60).

    ______________________________________                                        (9)    HPLC analysis                                                          ______________________________________                                        Column:         Cosmosil 5C.sub.18 -AR, 5 μm,                                              4.6 mm I.D. × 150 mm                                    Eluent:         CH.sub.3 CN: 0.02M phosphate buffer,                                          pH 7.0 (15:85)                                                Flow rate:      1.0 ml/min.                                                   UV detector:    254 nm                                                        Retention time: 11.5 min.                                                     Internal standard:                                                                            Pradimicin A (Rt = 9.7 min.)                                  ______________________________________                                    

(10) ¹ H NMR spectrum: as shown in FIG. 2

(11) ¹³ C NMR spectrum: as shown in FIG. 3

Desxylosyl BMY-28960 has the following physico-chemical properties:

(1) Appearance: Deep reddish orange powder

(2) Melting point: >180° C.

(3) HR-FAB-MS (positive): m/z 712.1871 (M+H)⁺

(4) Molecular formula: C₃₄ H₃₃ NO₁₆

(5) UV absorption spectrum, λmax nm(δ): in H₂ O-MeOH-DMSO (4.5:4.5:1):470(10,100), in 0.02 N HCl-MeOH-DMSO (4.5:4.5:1): 461 (10,600) in 0.02 NNaOH-MeOH (1:1): 213(31,500), 242(30,100) 320(13,600), 498(12,600).

(6) IR spectrum (KBr) ν_(max) cm⁻¹ : as shown in FIG. 4

(7) Solubility in solvent

Soluble: Dimethyl sulfoxide, dimethylformamide, and alkaline water

Slightly soluble: Ethanol, methanol, acetone and acetonitrile

Isoluble: Acidic water, n-butanol, ethyl acetate, chloroform, and othercommon organic solvents

(8) ¹ H NMR spectrum: as shown in FIG. 5.

VI. Biological Activities of the Antibiotic

The in vitro antifungal activities of BMY-28960 and desxylosyl BMY-28960obtained by the present fermentation process were determined by aconventional agar dilution method on yeast morphology agar containing1/15 M phosphate buffer (pH 7.0). The results are shown in Tables 4 and5.

                  TABLE 4                                                         ______________________________________                                        In vitro antifungal activity of BMY-28960                                                        BMY-    Ampho-   Keto-                                     Test organism      28960   tericin B                                                                              canazole                                  ______________________________________                                        Saccharomyces cerevisiae                                                                         3.1     0.4      100                                       ATCC 9763                                                                     Candida albicans A9540                                                                           6.3     0.4      25                                        Candida albicans ATCC 38247                                                                      1.6     6.3      6.3                                       Candida albicans ATCC 32354                                                                      3.1     0.2      50                                        (B311)                                                                        Candida albicans 83-2-14 (Juntendo)                                                              12.5    0.4      25                                        Candida tropicalis 85-8 (Kitasato)                                                               12.5    0.4      100                                       Candida tropicalis IFO 10241                                                                     3.1     0.4      50                                        Cryptococcus neoformans D49                                                                      3.1     0.4      6.3                                       Cryptococcus neoformans IAM 4514                                                                 6.3     0.4      6.3                                       Aspergillus fumigatus IAM 2034                                                                   6.3     0.4      3.1                                       Trichophyton mentagrophytes #4329                                                                6.3     0.4      0.8                                       ______________________________________                                         Medium: Yeast morphology agar + 1/15M phosphate buffer (pH 7.0)               Inoculum size: 10.sup.6 cells/ml (10.sup.7 cells/ml for T. mentagrophytes     Incubation conditions: 28° C., 40 hrs. (60 hrs. for T.                 mentagrophytes)                                                          

                  TABLE 5                                                         ______________________________________                                        In vitro antifungal activity of desxylosyl BMY-28960                                            Desxylosyl                                                                              BMY-    Ampho-                                    Test organism     BMY-28960 28960   tericin B                                 ______________________________________                                        Saccharomyces cerevisiae                                                                        3.1       1.6     0.8                                       ATCC 9763                                                                     Candida albicans A9540                                                                          3.1       3.1     0.8                                       Candida albicans IAM 4888                                                                       3.1       3.1     0.8                                       Candida albicans ATCC 32354                                                                     3.1       3.1     0.8                                       (B311)                                                                        Candida albicans 83-2-14                                                                        1.6       3.1     0.8                                       (Juntendo)                                                                    Candida tropicalis 85-8 (Kitasato)                                                              1.6       12.5    1.6                                       Candida tropicalis IFO 10241                                                                    6.3       12.5    1.6                                       Cryptococcus neoformans D49                                                                     1.6       3.1     0.8                                       Cryptococcus sp. IAM 4514                                                                       3.1       1.6     0.8                                       Aspergillus fumigatus IAM 2034                                                                  6.3       3.1     0.8                                       Trichophyton mentagrophytes                                                                     6.3       3.1     1.6                                       #4329                                                                         ______________________________________                                         Medium: Yeast morphology agar + 1/15M phosphate buffer (pH 7.0)               Inoculum size: 10.sup.6 cells/ml (Tm: 10.sup.7 cells/ml)                      Incubation conditions: 28° C., 40 hrs. (60 hrs. for T.                 mentagrophytes)                                                          

The in vivo efficacy of BMY-28960 was evaluated against Candida albicansA9540 systemic infection in male ICR mice (20-24 g body weight). Fivemice are used for each dose level. Mice were challenged intravenouslywith 10 times the median lethal dose of the pathogen suspended in salineand BMY-28960 was intravenously administered once immediately after thechallenge. The median protective dose (PD₅₀) was calculated fromsurvival rates recorded on day 21. The results are summarized in Table6.

                  TABLE 6                                                         ______________________________________                                        In vivo efficacy against C. albicans A9540                                    systemic infection in mice                                                    Compound       PD.sub.50 (mg/kg, i.v.)                                        ______________________________________                                        BMY-28960      6.7                                                            Pradimicin A   10                                                             Amphotericin B 0.31                                                           Fluconazole    >50                                                            ______________________________________                                    

BMY-28960 was well-tolerated in ICR mice; neither lethality nor apparentside effects were noted following intravenous administration ofBMY-28960 up to 600 mg/kg.

The following examples are provided in order to more fully illustratethe present invention, and they shall not be construed as in any mannerlimiting the scope of the invention.

EXAMPLE 1 Seed culture

A stock culture of the producing organism, Actinomadura sp. AB 1236,(ATCC 55208) was streaked on YSM agar slant and incubated at 37° C. for2 weeks. One loopful of the strain was inoculated into a 500-mlErlenmeyer flask containing 100 ml of a medium composed of glucose 0.5%,soluble starch 2%, yeast extract 0.2%, NZ-case 0.3%, fish meal extractD30X (Banyu Eiyou K.K.) 0.5% and CaCO₃ 0.3% (the pH of the medium wasadjusted to 7.0 before autoclaving). The culture was incubated on arotary shaker at 32° C. for 5 days and was used as the seed culture.

EXAMPLE 2 Flask fermentation

Each 5-ml portion of the seed culture as set forth in Example 1 wastransferred into a 500-ml Erlenmeyer flask containing 100 ml ofproduction medium composed of glycerol 2%, Esusan mi-to (soybean meal;Ajinomoto Co., Inc.) 1.5%, K₂ HPO₄ 0.0025%, KH₂ PO₄ 0.1125%, CoCl₂.6H₂ O0.0005% and D-serine 0.125% with D-cycloserine (Wako Pure ChemicalIndustries Ltd.) 10 μg/ml. The culture was incubated on a rotary shakeroperating at 200 rpm and 28° C. for 12 days, at which time production ofBMY-28960 reached 530 μg/ml.

EXAMPLE 3 Flask fermentation (effect of added threonine)

Each 5-ml. portin of the seed culture as set forth in Example 1 wastransferred into a 500-ml Erlenmeyer flask containing 100 ml ofproduction medium composed of glycerol 2%, Pharmamedia (Traders Protein)1.5%, KH₂ PO₄ 0.1%, L- or D-threonine 0.2%, CoCl₂ 6H₂ O 0.0005%,D-serine 0.2% and D-cycloserine 10 μg/ml. The culture was incubated on arotary shaker operating at 200 rpm for 11 days at 28° C. To see theeffect of threonine on the production of BMY-28960, fermentation wasalso carried out in the absence of threonine. The results are summarizedbelow:

    ______________________________________                                        Amino acid       Production of BMY-28960                                      ______________________________________                                        L-Threonine      1008 μg/ml                                                D-Threonine       954                                                           --              780                                                         ______________________________________                                    

EXAMPLE 4 Isolation and purification of BMY-28960

The fermentation broth (2.0 liter) of Example 2 was acidified to pH 2.0using 6 N HCl and centrifuged. The supernatant (1.7 liter) was appliedon a column of Diaion HP-20 (300 ml), and the column was first washedwith water (1.5 liter) and then eluted with 1.6 liter of methanol. Theeluate was concentrated and then lyophilized to yield 2.1 g of a crudesolid. This solid was dissolved in 1 liter of water, and the pH of thesolution was adjusted to 6.3 with 1 N NaOH. The solution was applied ona column of Diaion HP-20, and the column was first washed with a mixtureof 0.002 N HCl:acetone (1:1) and then eluted with 1 liter of a mixtureof 0.002 N NaOH:acetone (1:1). Concentration of the eluate afforded asolid (690 mg). A part (400 mg) of the solid was dissolved inacetonitrile:0.02 M phosphate buffer, pH 7.0 (12.5:87.5, 30 ml) andsubjected to chromatography on a column of YMC gel, ODS A60 (500 ml,Yamamura Chemical Lab.) which had been equilibrated with the samesolvent system. Elution was done with the same solvent. The fractionscontaining BMY-28960 were concentrated, desalted by Diaion HP-20 andlyophilized to give BMY-28960 (82 mg).

EXAMPLE 5 Isolation and purification of desxylosyl BMY-28960

The culture broth (25 L) prepared by fermentation of strain AB1236 inthe presence of D-serine was centrifuged. The supernatant (27 L) waspassed through 3.8 L of Diaion HP-20. The resin was washed successivelywith 80% aq. acetone (8 L) and acetone-0.01 N HCL (60:40) mixture (12L), and then eluted with acetone-0.01 N NaOH (60:40) mixture (12 L) toyield 16.04 g of crude. The complex (2 g) was dissolved in 200 ml of amixture of CH₃ CN-0.02 M phosphate buffer, pH 7.0 (13.87) and charged ona column of YMC gel, ODS-A60 (10 L, Yamamura Chemical Lab.) which hadbeen equilibrated with the same solvent system. Elution was carried outwith the solvent system used to dissolve the sample and the eluates weremonitored by HPLC (Column: Cosmosil 5C₁₈ AR, 5 μm, 4.6 mm i.d.×150 mm,Nacalai Tesque Inc., Mobile phase: CH₃ CN-1/15 M phosphate buffer, pH3.5 (27:73), Flow rate: 1.0 ml/min., Detection: UV absorption at 254 nm,Retention time: desxylosyl BMY-28960 8.3 min.: BMY-28960, 7.6 min.). Thefaster eluted red fraction (3.7 L) containing desxylosyl BMY-28960 wasconcentrated, and chromatographed on a column of Diadon HP-20 (30 ml)using acetone- 0.1 N NaOH (60:40) mixture (50 ml) as eluent. The redeluates were concentrated and lyophilized to afford 29 mg of solid. Anaqueous solution (30 ml) of the sample was adjusted to pH 2.5 with 0.1 NHCl to deposit pure desxylosyl BMY-28960 (19 mg). The slower elutedfraction (24 L) from YMC gel chromatography was similarly worked up toafford 700 mg of pure BMY-28960.

We claim:
 1. A process for the preparation of an antibiotic of theformula ##STR3## wherein R is hydrogen or β-D-xylosyl, which comprisescultivating a strain of Actinomadura capable of producing saidantibiotic in an aqueous medium containing an assimilable source ofcarbon, nitrogen, and D-serine under aerobic condition, and recoveringsaid antibiotic from the cultured broth; said Actinomadura strain beingthe strain designated AB 1236 and deposited with the American TypeCulture Collection under the Accession No. ATCC
 55208. 2. A processaccording to claim 1 wherein said medium further contains D-cycloserine.3. A process according to claim 2 wherein said medium further containsthreonine.